Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilm-associated infections on the pulmonary secretions of patients with the genetic disorder cystic fibrosis (CF). These infections are often impossible to eradicate with traditional antibiotic therapies. Therefore, investigators have performed global transcriptional and proteomics studies on P. aeruginosa to identify new molecular targets that are induced in P. aeruginosa during infectious processes. In many cases, production of specific proteins is regulated at the post-transcriptional level by small trans-acting RNAs or by cis-acting RNAs associated with the 5'-untranslated region of the mRNA. These non-protein-coding RNAs (ncRNAs) control expression of many cellular processes, including those involved in quorum sensing and virulence factor production. Approximately twenty P. aeruginosa ncRNAs have been identified previously. Based on our bioinformatics studies, this number is only a fraction of those actually encoded by P. aeruginosa. We predict that over 100 ncRNAs are encoded in the intergenic regions of the P. aeruginosa genome, and that others may be misannotated as protein-coding sequence. We have demonstrated expression of many of these predicted ncRNAs by using custom-designed microarrays and by Northern blotting experiments. Our present goals are to: (I) Map the genome position of the P. aeruginosa ncRNA, and to represent each on a custom microarray with tiled probes. Expression of these ncRNAs will be determined by using microarrays, Northern blotting, and mutational studies. (II) Define the biological activities of a novel ncRNA, located between P. aeruginosa genes PA4634 and PA4635. This small ncRNA is expressed only during stationary phase when P. aeruginosa is starved for iron, conditions likely to exist in the interiors of infectious biofilms. We will characterize the expression of this ncRNA and identify its mRNA targets. Our long-term goal is to identify new molecular targets that can be used for anti-P. aeruginosa therapies. These studies will facilitate this goal by identifying and characterizing many new regulatory molecules important in P. aeruginosa pathogenesis. PUBLIC HEALTH RELEVANCE: The opportunistic pathogen, Pseudomonas aeruginosa, colonizes pulmonary tissue of patients with the genetic disorder, cystic fibrosis. These infections are often resistant to antibiotic treatment. The goals of this study are to define post- transcriptional regulatory processes that occur during P. aeruginosa infection, in order to define novel molecular targets for anti-P. aeruginosa therapies.